Micromolded or 3-D printed pulsatile release vaccine formulations

ABSTRACT

Emulsion-based and micromolded (“MM”) or three dimensional printed (“3DP”) polymeric formulations for single injection of antigen, preferably releasing at two or more time periods, have been developed. Formulations are preferably formed of biocompatible, biodegradable polymers. Discrete regions encapsulating antigen, alone or in combination with other antigens, adjuvants, stabilizers, and release modifiers, are present in the formulations. Antigen is preferably present in excipient at the time of administration, or on the surface of the formulation, for immediate release, and incorporated within the formulation for release at ten to 45 days after initial release of antigen, optionally at ten to 90 day intervals for release of antigen in one or more additional time periods. Antigen may be stabilized through the use of stabilizing agents such as trehalose glass. In a preferred embodiment for immunization against polio, antigen is released at the time of administration, and two, four and six months thereafter.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/916,555, filed Dec. 16, 2013. Application No. 61/916,555, filed Dec.16, 2013, is hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention is generally in the field of injectable vaccineformulations providing multiple releases of vaccine.

BACKGROUND OF THE INVENTION

Vaccines typically involve an initial dose of antigen, followed by oneor more booster doses at defined times after the initial administration,typically ten to 60 days later. The need for administration of a boosterdose clearly limits the practicality of vaccines in much of the world,as well as increases costs and difficulties in agriculturalapplications.

Polymeric microspheres have the potential to be effective vaccinedelivery vehicles. They have the ability to enhance targeting of antigenpresenting cells (APCs) and have the potential for controlled, sustainedrelease of antigen-thereby potentially eliminating the need for multiplevaccination doses. Further, the polymer matrix can act as a shield froma hostile external environment and has the potential to reduce adversereactions and abrogate problems caused by the vaccine strain inimmunocompromised individuals. PLGA microspheres have been developed forsingle immunization, with and without burst release. Given thebiodegradable nature and sustained release properties that PLGA offers,microspheres formulated from PLGA could be useful for the delivery ofvaccines. Summarized in Kirby et al., Chapter 13: Formation andCharacterisiation of polylactide-co-galactide PLGA microspheres (2013).PLGA based microparticles are traditionally produced by doubleemulsion-solvent evaporation, nano-precipitation, cross-flow filtration,salting-out techniques, emulsion-diffusion methods, jet milling, andspray drying. Summarized in Kirby et al., Chapter 13: Formation andCharacterisiation of polylactide-co-galactide PLGA microspheres (2013).PLGA microspheres can also be formulated to incorporate a range ofmoieties, including drugs and proteins, that can act as adjuvants. Ithas been contemplated that PGLA particles produced by these methods canbe lyophilized and stored for later use and delivery.

Hanes et al., Adv. Drug. Del. Rev., 28: 97-119 (1997), report onattempts to make polymeric microspheres to deliver subunit protein andpeptide antigens in their native form in a continuous or pulsatilefashion for periods of weeks to months with reliable and reproduciblekinetics, to obviate the need for booster immunizations. Microsphereshave potential as carriers for oral vaccine delivery due to theirprotective effects on encapsulated antigens and their ability to betaken up by the Peyer's patches in the intestine. The potency of theseoptimal depot formulations for antigen may be enhanced by theco-delivery of vaccine adjuvants, including cytokines, that are eitherentrapped in the polymer matrix or, alternatively, incorporated into thebackbone of the polymer itself and released concomitantly with antigenas the polymer degrades.

As reported by Cleland et al., J. Controlled Rel. 47(2):135-150 (1997),the administration of a subunit vaccine (e.g., gp120) for acquiredimmunodeficiency syndrome (AIDS) can be facilitated by a single shotvaccine that mimics repeated immunizations. Poly(lactic-co-glycolicacid) (PLGA) microspheres were made that provide a pulsatile release ofgp120. Microspheres were made using a water-in-oil-in-watermicroencapsulation process with either methylene chloride or ethylacetate as the polymer solvent. The protein was released underphysiological conditions in two discrete phases: an initial burstreleased over the first day and after several weeks or months, a secondburst of protein was released. The second burst of protein was dependentupon the PLGA inherent viscosity and lactide/glycolide ratio (bulkerosion).

These studies demonstrate that it is possible to achieve a vaccineresponse using injectable microparticles. However, no such product hasever been approved for human or animal use. It is difficult to achieveeffective loading of antigen, uniformity of encapsulation and release,and extremely low levels of solvent not affecting antigenicity.

It is estimated that precluding the need for a “cold chain” for vaccinedistribution through the development of thermo-stable formulations couldsave about $200 million annually. Trouble with implementing thesestrategies rests on the lack of appropriate cryprotectant methods. ASummarized in Kirby et al., Chapter 13: Formation and Characterisiationof polylactide-co-galactide PLGA microspheres (2013). Similarinformation and disclosure on nanovaccines can be found in Gregory etal., Frontiers in Cell and Infect. Microbio. 3:Article 13 (2013).Nandedkar, J. Biosci. 34:995-1003 (2009). Stabilization of proteinsincluded in microspheres is problematic. A number of types ofstabilizing excipients have been studied. Summarized in Kim and Pack,BioMEMS and Biomedical Nanotechnology. 1:19-50 (2006). Additionally, thetype of polymer used for microsphere fabrication, its degradation rate,acidity of the degradation products, hydrophobicity, etc., can alsoimpact the stability of incorporated proteins.

It is therefore an object of the present invention to provide injectablepolymeric formulations providing release of encapsulated antigen at twoor more times.

It is a further object of the present invention to provide injectablepolymeric formulations which do not damage and which can stabilizeencapsulated antigen.

It is a still further object of the present invention to provide methodsand materials for micromolding and three-dimensional printing ofinjectable polymeric formulations providing release of encapsulatedantigen at two or more times, and the resulting formulations.

SUMMARY OF THE INVENTION

Emulsion-based and Micromolded (“MM”) or three dimensional printed(“3DP”) polymeric formulations for single injection of antigen,preferably releasing at two or more time periods, have been developed.Formulations are preferably formed of biocompatible, biodegradablepolymers. Discrete regions encapsulating antigen, alone or incombination with other antigens, adjuvants, stabilizers, and releasemodifiers, are present in the formulations. Antigen is preferablypresent in excipient at the time of administration, or on the surface ofthe formulation, for immediate release, and incorporated within theformulation for release at ten to 45 days after initial release ofantigen, optionally at ten to 90 day intervals for release of antigen inone or more additional time periods. Antigen may be stabilized throughthe use of stabilizing agents such as trehalose glass. In a preferredembodiment for immunization against polio, antigen is released at thetime of administration, and two, four and six months thereafter. In apreferred embodiment, leakage between bursts of release is minimal andrelease occurs over a narrow time frame.

Studies demonstrate the selection of polymer and solvent systems thatprovides for discrete release of antigen, without overlap, with minimaldegradation or damage to the encapsulated antigen. Preferred solventsinclude methylene chloride and chloroform, and preferred polymers arepolylactic acid (“PLA”), polyglycolic acid (“PGA”), and copolymersthereof (“PLGA”).

Formulations are designed for subcutaneous or intramuscular injectionvia needle or cannula, for topical injection to a mucosal region such asintranasal, or by scarification to the epidermis. Preferred applicationsare for administration of antigen eliciting an effective immune responseto infectious agents such as bacteria, virus, protozoan and parasiticorganisms. However, formulations may also be used for administration ofother therapeutic, prophylactic or diagnostic agents, alone or incombination with antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the effect of lowering excipient:vaccineratio on percentage of D-antigen retained after drying 26× Trivalent IPVon PLA for 16 hours at room temperature and humidity. Excipients: 10%sorbitol, 8.5% MSG, 8.5% MgCl₂.

FIG. 2 is a line graph showing long-term stability of lyophilized IPV(type I, type II, and type III) with excipients at 37° C. in humidifiedatmosphere. The stability is expressed as percent recover (% Recovery)over time (days). On the line graph, the line for type I IPV isdesignated (1), the line for type II IPV is designated (2), and the linefor type III IPV is designated (3).

FIGS. 3A and 3B are graphs of the release of a model protein, bovineserum albumin (“BSA”) (FIG. 3A, micrograms/10 mg of microspheres; FIG.3B, % BSA) over time (weeks) for 5% BSA and 0.5% BSA from PLLA, 50 kD.

FIGS. 4A and 4B are graphs of the release of a model protein, bovineserum albumin (“BSA”) (FIG. 4A, micrograms/10 mg of microspheres; FIG.4B, % BSA) over time (weeks) for 5% BSA and 0.5% BSA from PLLA, 100 kD.

FIGS. 5A and 5B are graphs of the release of a model protein, bovineserum albumin (“BSA”) (FIG. 5A, micrograms/10 mg of microspheres; FIG.5B, % BSA) over time (weeks) for 5% BSA and 0.5% BSA from PLLA, 300 kD.

FIGS. 6A and 6B are graphs of the release of a model protein, bovineserum albumin (“BSA”) (FIG. 6A, micrograms/10 mg of microspheres; FIG.6B, % BSA) over time (weeks) for 5% BSA and 0.5% BSA from P(d,l)LA, 20kD.

FIGS. 7A and 7B are graphs of protein release over time (weeks) for PLGAformulations (FIG. 7A) and bolus injections (FIG. 7B).

FIGS. 8A and 8B are graphs of the release of 0.5% of a model protein,bovine serum albumin (“BSA”) compared to release of 0.5% of anothermodel protein ovalbumin (“OVA”) (FIG. 8A, micrograms/10 mg ofmicrospheres; FIG. 8B, % BSA) over time (weeks) from PLGA (50:50), 20kD, derivatized with a carboxylic group.

FIGS. 9A and 9B are graphs of the release of 5% of a model protein,bovine serum albumin (“BSA”) compared to release of 5% of another modelprotein ovalbumin (“OVA”) (FIG. 9A, micrograms/10 mg of microspheres;FIG. 9B, % BSA) over time (weeks) from PLGA (50:50), 31 kD, derivatizedwith a carboxylic group.

FIGS. 10A and 10B are graphs of the release of 0.5% of a model protein,bovine serum albumin (“BSA”) compared to release of 0.5% of anothermodel protein ovalbumin (“OVA”) (FIG. 10A, micrograms/10 mg ofmicrospheres; FIG. 10B, % BSA) over time (weeks) from PLGA (50:50), 31kD, derivatized with a carboxylic group.

FIGS. 11A and 11B are schematics of particles made by emulsion (FIG.11A) and by 3D printing or micromolding (FIG. 11B), and the release ofprotein from the emulsion particle (FIG. 11C) and the 3DP particle (FIG.11D), which models the release of protein from bolus injections. FIG.11E is a schematic of polymer degradation-based bursts with one monthlag to simulate bolus injections.

FIGS. 12A-12D are schematics of the 3D printing process: Creating thestructure of PLGA particles (FIG. 12A), filling drugs or proteins intothe particles (FIG. 12B), drying the drugs or proteins (FIG. 12C), andencapsulation of the particles (FIG. 12D).

FIG. 13 is a schematic of the waveform parameters to be optimized toproduce uniform single droplets of “ink” (polymer solution) duringprinting from a piezoelectric nozzle jet.

FIGS. 14A-14C are graphs of the percent D-antigen (IPV type I, II, andIII) retained after lyophilizing with sugar excipients 1 M trehalose, 1M sucrose, and 3 M sucrose, then incubating at 4° C., 25° C. or 37° C.

FIGS. 15A and 15B are graphs of the percent D-antigen (IPV type I, II,and III) retained after lyophilizing with 0, 0.5, 0.75, 1 or 1.25 Mtrehalose, 1 then incubating at 4° C. (FIG. 15A) or 25° C. (FIG. 15B).

FIGS. 16A and 16B are graphs of the percent D-antigen (IPV type I, II,and III) retained after mixing with solvents until solvent evaporation,with solvents tetrafluoroethylene (“TFE”), dichloromethane (“DCM”), orTFE and DCM, without sugar excipient (FIG. 16A) or with 0.75 M trehaloseas excipient (FIG. 16B).

FIG. 17A is a graph of the percent D-antigen (IPV type I, II, and III)retained after IVP with 0 M sugar, 0.5 M trehalose, or 0.5 Mtrehalose-sucrose is printed onto 3D-printed PLA substrate and dried at25° C., 22% RH. FIG. 17B is a graph of the percent D-antigen (IPV typeI, II, and III) retained after IVP with 0.25 M trehalose or 0.5 Mtrehalose-sucrose is printed onto 3D-printed PLA substrate and dried at25° C., 10.7% RH.

FIGS. 18A and 18B are graphs of the percent D-antigen retained afterlyophilization with 0.5 or 0.75 M trehalose then incubating with 25° C.(FIG. 18A) or after lyophilization with 0.5 M trehalose-sucrose,pipetted, or 0.5 M trehalose-sucrose, jetted, then drying at 25° C.,10.7% RH (FIG. 18B).

FIGS. 19A-19C are graphs showing anti-BSA IgG (antibody) titers (in log2 values) plotted for the groups presented in Table 3 at 1 week (FIG.19A), 2 weeks (FIG. 19B), and 4 weeks (FIG. 19C) post-injection. Thenegative controls are all zero and the microsphere formulations aregenerating an equivalent or stronger response compared to the boluscontrol. (F4 and F8 are blank microspheres, no drug; see Table 4).

FIGS. 20A-20C are histograms showing size distribution of microspheresprepared with formulation C (FIG. 20A), formulation G (FIG. 20B), andformulation E (FIG. 20C).

FIGS. 20D-20F are histograms showing volume distribution of formulationC (FIG. 20D), formulation G (FIG. 20E), and formulation E (FIG. 20F).

FIGS. 21A-21D are line graphs showing weekly BSA release (in percentages(%)) from microspheres formulated with 7-17 kDa, 50:50 PLGA (FIG. 21A),24-38 kDa, 50:50 PLGA (FIG. 21B), 38-54 kDa, 50:50 PLGA (FIG. 21C), and4-15 kDa, 75:25 PLGA (FIG. 21D) containing 0.5, 3, or 5% BSA by weight.Error bars represent standard deviation.

FIGS. 22A and 22B are line graphs showing IgG antibody titers in mousesera against BSA released from microparticles and plotted as geometricmean over time on a log 2 scale. FIG. 22A shows low-dose formulations C& G compared to a series of three dose-matched bolus BSA injections.FIG. 22B shows high-dose formulation E compared to its dose-matchedbolus control. Bolus BSA was injected at 0, 4, and 8 weeks in thecontrol groups. Filled symbols indicate significant differences betweenthe group with unfilled symbols of the same shape and dose-matchedcontrol at that time point determined using ANOVA analysis for A (3groups) and Student's t-test for B (2 groups). One, two, and threefilled symbols indicate p<0.05, p<0.01, and p<0.001 respectively anderror bars represent standard deviation.

FIGS. 23A and 23B are dot plots showing peak titers induced byimmunization at low (FIG. 23A), and high (FIG. 23B) BSA dosing. Peaktiters for all microparticle formulations occurred at week 4 while bothbolus injection groups peaked at week 10. NS represents not significantwhen using a Student's t-test with Holm-Bonferroni correction formultiple comparisons at a significance level of 0.05. Adjusted p valuesfor formulations C, G, and E are 0.0645, 0.0543, and 0.0784respectively.

FIGS. 24A-24C are schematics of the micromolding process showing thesteps of shell microfabrication (FIG. 24A), the filling of shells withdrug (FIG. 24B), and sealing the shell with a cap (FIG. 24C).

FIG. 25 is a bar graph showing the stability of IPV (% Recovery) with orwithout excipients gelatin, maltodextrin, pullulan, myristic acid, andTween80, during organic/aqueous mixing by vortexing or sonication.

FIG. 26 is a bar graph showing the stability of IPV (% Recovery) with orwithout excipients gelatin, maltodextrin, pullulan, myristic acid, andTween80, during organic/aqueous mixing with PLGA by vortexing orsonication (son=sonication).

FIG. 27 is a bar graph showing the stability of IPV (% Recovery) withexcipients pullulan, pullulan/BSA, sorbitol/MSG/MgCl2, following dryingby overnight lyophylization, or using Genevac for 1 h at 30-35° C.

FIG. 28 is a line graph showing change in pH of release medium by PLGAparticles over time (days) in the presence of buffering agents Al(OH)₃(line (2)), myristic acid (line (3)), and Mg(OH)₂ (line (4)). The linerepresenting change in pH of release medium over time in the absence ofbuffering agents is labeled (1).

FIG. 29 is a line graph showing change in pH of release medium by PLGAparticles over time (days). The line for PLGA 502H tested afterreplacing buffer every 2-3 days is designated (1), the line for PLGA502H tested after replacing buffer every 7 days is designated (2), theline for PLGA 503H tested after replacing buffer every 2-3 days isdesignated (3), and the line for PLGA 503H tested after replacing bufferevery 7 days is designated (4).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Additive manufacturing” or “3D printing” as used herein refers to aprocess of making a three-dimensional solid object of virtually anyshape from a digital model. 3D printing is achieved using an additiveprocess, where successive layers of material are laid down in differentshapes or thicknesses. In some embodiments, “3D printing” involves anextruded or solvent based polymer-containing ink (e.g., PLGA, PLLA,etc.) that is jetted or extruded through a nozzle and solidified into adesired shape. The shape can be controlled in the x, y and z directions.

“Micromolding,” as used herein, generally refers to processes suitablefor manufacturing parts or devices on a microscale, or processessuitable for manufacturing parts or devices having features ortolerances on a microscale. Exemplary techniques include, but are notlimited to, lithography.

“Microdevice,” as used herein, refers to any object or device havingmicroscale dimensions, such as 1 micron to 1000 microns, 1 micron to 500microns, 1 micron to 250 microns, or 1 micron to 100 microns.

The term “diameter” is art-recognized and is used herein to refer toeither of the physical diameter or the hydrodynamic diameter. Thediameter of emulsion typically refers to the hydrodynamic diameter. Thediameter of the capsules, both in spherical or non-spherical shape, mayrefer to the physical diameter in the hydrated state. The diameter ofthe particles, colloids and cells which are encapsulated inside thecapsules refers to the physical diameter in the hydrated state. As usedherein, the diameter of a non-spherical particle or a non-sphericalcapsule may refer to the largest linear distance between two points onthe surface of the particle. When referring to multiple particles orcapsules, the diameter of the particles or the capsules typically refersto the average diameter of the particles or the capsules. Diameter ofparticles or colloids can be measured using a variety of techniques,including but not limited to the optical or electron microscopy, as wellas dynamic light scattering.

The term “biocompatible” as used herein refers to one or more materialsthat are neither themselves toxic to the host (e.g., an animal orhuman), nor degrade (if the material degrades) at a rate that producesmonomeric or oligomeric subunits or other byproducts at toxicconcentrations in the host.

The term “biodegradable” as used herein means that the materialsdegrades or breaks down into its component subunits, or digestion, e.g.,by a biochemical process, of the material into smaller (e.g.,non-polymeric) subunits.

The term “microspheres,” “microparticles,” or “microcapsules” isart-recognized, and includes substantially spherical solid or semi-solidstructures, e.g., formed from biocompatible polymers such as subjectcompositions, having a size ranging from about one or greater up toabout 1000 microns, such as 1 micron to 500 microns, 1 micron to 250microns, or 1 micron to 100 microns. The term “microparticles” is alsoart-recognized, and includes microspheres and microcapsules, as well asstructures that may not be readily placed into either of the above twocategories, all with dimensions on average of less than about 1000microns. A microparticle may be spherical or nonspherical and may haveany regular or irregular shape. If the structures are less than aboutone micron in diameter, then the corresponding art-recognized terms“nanosphere,” “nanocapsule,” and “nanoparticle” may be utilized.

“Narrow range of release,” as used herein, generally means that theagent is released over a specific period of time, such as an hour,hours, a day, a week, a month, etc.)

“Antigen” or “Vaccine,” as used herein, refers to any molecule or entitythat produces a specific immune response in a host organism, such as amammal.

“Immune response,” as used herein, refers to a specific response to anantigen or vaccine that produces immunity to any future exposure in ahost, such as a mammal.

“Water soluble” generally refers to something that dissolves or comesapart in aqueous environment.

“Emulsion” as used herein refers to a liquid discrete phasehomeogeneously dispersed in a liquid continuous phase.

“Hydrophilic,” as used herein, refers to molecules which have a greateraffinity for, and thus solubility in, water as compared to organicsolvents. The hydrophilicity of a compound can be quantified bymeasuring its partition coefficient between water (or a buffered aqueoussolution) and a water-immiscible organic solvent, such as octanol, ethylacetate, methylene chloride, or methyl tert-butyl ether. If afterequilibration a greater concentration of the compound is present in thewater than in the organic solvent, then the compound is consideredhydrophilic.

“Hydrophobic,” as used herein, refers to molecules which have a greateraffinity for, and thus solubility in, organic solvents as compared towater. The hydrophobicity of a compound can be quantified by measuringits partition coefficient between water (or a buffered aqueous solution)and a water-immiscible organic solvent, such as octanol, ethyl acetate,methylene chloride, or methyl tert-butyl ether. If after equilibration agreater concentration of the compound is present in the organic solventthan in the water, then the compound is considered hydrophobic.

II. Formulations

A. Polymers and Solvent Systems

Polymers

The formulations, which may be formed of microparticles, includingmicrospheres or microcapsules and including those that areemulsion-based, or devices such as those prepared by micromolding, areformed of polymers. Antigen may be dispersed or encapsulated by thepolymer. In one embodiment, the device contains a core that onlycontains one or more vaccines or antigen and stabilizers and the shellor particle wall only contains one or more biodegradable polymers withor without additives. Polymer without antigen may be used to seal orseparate areas of the formulation from other areas, and release atdifferent rates.

Polymers must be biocompatible and processible under conditions andusing reagents that preserve the antigen. The formulation can be madewith hydrophilic polymers, hydrophobic polymers, amphiphilic polymers,or mixtures thereof. The formulation can contain one or more hydrophilicpolymers.

Hydrophilic polymers include cellulosic polymers such as starch andpolysaccharides; hydrophilic polypeptides; poly(amino acids) such aspoly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-asparticacid, poly-L-serine, or poly-L-lysine; polyalkylene glycols andpolyalkylene oxides such as polyethylene glycol (PEG), polypropyleneglycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyol);poly(olefinic alcohol); polyvinylpyrrolidone);poly(hydroxyalkylmethacrylamide); poly(hydroxyalkylmethacrylate);poly(saccharides); poly(hydroxy acids); poly(vinyl alcohol), andcopolymers thereof.

Examples of hydrophobic polymers include polyhydroxyacids such aspoly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolicacids); polyhydroxyalkanoates such as poly3-hydroxybutyrate orpoly4-hydroxybutyrate; polycaprolactones; poly(orthoesters);polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones);polycarbonates such as tyrosine polycarbonates; polyamides (includingsynthetic and natural polyamides), polypeptides, and poly(amino acids);polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates);hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals;polycyanoacrylates; polyacrylates; polymethylmethacrylates;polysiloxanes; poly(oxyethylene)/poly(oxypropylene) copolymers;polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates;polyalkylene succinates; poly(maleic acids), as well as copolymersthereof. In certain embodiments, the hydrophobic polymer is an aliphaticpolyester. In preferred embodiments, the hydrophobic polymer ispoly(lactic acid), poly(glycolic acid), or poly(lactic acid-co-glycolicacid).

The formulation can contain one or more biodegradable polymers.Biodegradable polymers can include polymers that are insoluble orsparingly soluble in water that are converted chemically orenzymatically in the body into water-soluble materials. Biodegradablepolymers can include soluble polymers crosslinked by hydolyzablecross-linking groups to render the crosslinked polymer insoluble orsparingly soluble in water.

Biodegradable polymers include polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetate phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexylmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinylchloride polystyrene and polyvinylpryrrolidone, derivatives thereof,linear and branched copolymers and block copolymers thereof, and blendsthereof. Exemplary biodegradable polymers include polyesters, poly(orthoesters), poly(ethylene imines), poly(caprolactones),poly(hydroxybutyrates), poly(hydroxyvalerates), polyanhydrides,poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates,polyphosphate esters, polyphosphazenes, derivatives thereof, linear andbranched copolymers and block copolymers thereof, and blends thereof.

Amphiphilic polymers can be polymers containing a hydrophobic polymerblock and a hydrophilic polymer block. The hydrophobic polymer block cancontain one or more of the hydrophobic polymers above or a derivative orcopolymer thereof. The hydrophilic polymer block can contain one or moreof the hydrophilic polymers above or a derivative or copolymer thereof.

In particularly preferred embodiments the microparticle containsbiodegradable polyesters or polyanhydrides such as poly(lactic acid),poly(glycolic acid), and poly(lactic-co-glycolic acid). Themicroparticles can contain one more of the following polyesters:homopolymers including glycolic acid units, referred to herein as “PGA,”and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, andpoly-D,L-lactide, collectively referred to herein as “PLA,” andcaprolactone units, such as poly(ε-caprolactone), collectively referredto herein as “PCL;” and copolymers including lactic acid and glycolicacid units, such as various forms of poly(lactic acid-co-glycolic acid)and poly(lactide-co-glycolide) characterized by the ratio of lacticacid:glycolic acid, collectively referred to herein as “PLGA;” andpolyacrylates, and derivatives thereof. Exemplary polymers also includecopolymers of polyethylene glycol (PEG) and the aforementionedpolyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers,collectively referred to herein as “PEGylated polymers.” In certainembodiments, the PEG region can be covalently associated with polymer toyield “PEGylated polymers” by a cleavable linker.

The formulation can contain one or a mixture of two or more polymers.The microparticles may contain other entities such as stabilizers,surfactants, or lipids.

Solvents

Solvents must be biocompatible, since some residue will always bepresent in the polymeric formulations. Representative polymer solventsinclude organic solvents such as chloroform, dichloromethane,tetrafluoroethylene, and acyl acetate. The antigen can be dissolved inaqueous or aqueous miscible solvents such as acetone, ethanol, methanol,isopropyl alcohol, and mixtures thereof.

B. Therapeutic, Prophylactic, Nutriceuticals, and Diagnostic Agents

Although described with reference to delivery of vaccines, it will beunderstood that the formulations may be used to provide release of avariety of therapeutic, prophylactic, nutriceutical, and/or diagnosticagents. These agents may be low molecular weight drugs, proteins such ashormones or growth factors, immunomodifiers, antibodies, nucleic acidmolecules (DNA, RNA, microRNA, siRNA).

Antigen

Infectious Agents

Antigens for delivery are killed or attenuated infectious agents such asbacteria such as Clostridia tetani, viruses such as hepatitis,influenza, and polio, and protozoans such as Plasmodium (malaria) andLeishmania. Table 2 lists some vaccines the antigens of which can beused in the disclosed formulations. Other antigens are antigenicproteins or haptens such as carbohydrate or sugar antigens effective asantigens for these infectious agents, as cancer antigens, or asimmunostimulants.

Poliomyelitis (Polio) is a highly contagious viral disease that invadesthe nervous system and can cause total paralysis in a matter of hours.One in 200 infections leads to irreversible paralysis, which is usuallyconfined to the legs. Among those paralyzed, 5% to 10% die due toparalysis of the diaphragm. There is no cure for polio. However, it canbe prevented by vaccination.

Polio cases have decreased by over 99% since 1988, from an estimated350,000 cases in more than 125 endemic countries to only 223 reportedcases in 2012. As of early 2013, only three countries (Afghanistan,Nigeria, and Pakistan) in the world were endemic for the disease.Despite aggressive vaccination efforts, polio has not been completelyeradicated and outbreaks still occur, particularly in developingcountries.

There are two types of vaccine that protect against polio: InactivatedPolio Vaccine (IPV) and Oral Polio Vaccine (OPV). To be effective, theIPV needs to be administered to the blood stream. In contrast, the OPVis effective by crossing intestinal epithelium. While the OPV conferssuperior intestinal immunity, is easy to administer, and is low in cost,live poliovirus is shed by the vaccinated. This is a concern where theentire population is not vaccinated.

As such, use of an IPV is preferable.

Cancer Antigens

Any protein produced in a tumor cell that has an abnormal structure dueto mutation can act as a tumor antigen. Such abnormal proteins areproduced due to mutation of the concerned gene. Mutation ofprotooncogenes and tumor suppressors which lead to abnormal proteinproduction are the cause of the tumor and thus such abnormal proteinsare called tumor-specific antigens. Examples of tumor-specific antigensinclude the abnormal products of ras and p53 genes. In contrast,mutation of other genes unrelated to the tumor formation may lead tosynthesis of abnormal proteins which are called tumor-associatedantigens.

Proteins that are normally produced in very low quantities but whoseproduction is dramatically increased in tumor cells, trigger an immuneresponse. An example of such a protein is the enzyme tyrosinase, whichis required for melanin production. Normally tyrosinase is produced inminute quantities but its levels are very much elevated in melanomacells.

Oncofetal antigens are another important class of tumor antigens.Examples are alphafetoproteins (AFP) and carcinoembryonic antigen (CEA).These proteins are normally produced in the early stages of embryonicdevelopment and disappear by the time the immune system is fullydeveloped. Thus self-tolerance does not develop against these antigens.

Abnormal proteins are also produced by cells infected with oncoviruses,e.g., Epstein Barr Virus (“EBV”) and Human Papillomavirus (“HPV”). Cellsinfected by these viruses contain latent viral DNA which is transcribedand the resulting protein produces an immune response. In addition toproteins, other substances like cell surface glycolipids andglycoproteins may also have an abnormal structure in tumor cells andcould thus be targets of the immune system.

Many tumor antigens have the potential to be effective as tumorvaccines. In addition to alpha fetoprotein (germ cell tumors,hepatocellular carcinoma) and carcinoembryonic antigen (bowel, lung,breast cancers), examples of tumor antigens include CA-125 (ovariancancer), MUC-1 (breast cancer, epithelial tumor antigen (breast cancer),and melanoma-associated antigen (malignant melanoma).

C. Stabilizing Agents

Antigen stability is defined as the maintenance of antigen structureduring formation of the vaccine formulation and at body temperature. Asdiscussed below, the polymer composition, selection of solvent, andprocessing conditions are critical to maintain antigen stability.

Stabilizing agents may also be added. Sugars are a typical group ofstabilizing agents for proteins. Examples include simple sugars such assucrose, fructose, mannitol, glucose, and trehalose as well as morecomplex sugars. See Alcock et al., Long-term thermostabilization of livepoxviral and adenoviral vaccine vectors at supraphysiologicaltemperatures in carbohydrate glass. Science Translational Medicine,2(19):19-19ra12 (2010).

Stabilization of the antigen can be determined by antigen specific ELISAin vitro and by measuring the immune response (e.g., IgGs) in animals invivo. Stability is evaluated during each step of the encapsulationand/or manufacturing process, during storage (at 25° C., room temp, highhumidity/high temp conditions, under physiological conditions (pH 7.2,37° C.) and in vivo (animal models).

D. Agents Increasing Rate or Completeness of Release

Gas-generated burst-release systems may allow for instantaneous releaseof encapsulated antigen. Pore forming agents which are removed byleaching or lyophilization may also be utilized.

III. Methods of Manufacture

It is essential that the methods used to manufacture the device maintainantigen stability, both during processing and at body temperature, andthat leakage following formation and administration are minimized.Post-formulation sterilization can typically be accomplished through acombination of sterile manufacturing conditions in combination withmethods such as gamma irradiation.

A. Emulsion

Microparticles can be made using standard techniques. A preferredtechnique is emulsification of a polymer solution in an organic solventwith an aqueous solution. Addition of organic phase to a large volume ofnon-solvent phase forms a spontaneous single emulsion and the resultingsolution is stirred continuously for solvent evaporation. Immediateformation of microspheres occurs. After stirring, microspheres arewashed and then dried.

The examples demonstrate formation of microparticles usingemulsification of polymer and antigen, alone or in combination withstabilizers such as trehalose and sucrose.

B. Three Dimensional Printing

3D printing could increase consistency of microspheres, allowing formore uniform release, as well as provide a means for making more complexdevices such as ‘Micro-rods’, having increased carrying capacity, thatcould eliminate the need for simultaneous release from multiplemicrospheres, as well as facilitate scale up.

Three-dimensional (3D) printing is a process of making 3D objects from adigital model. 3D printing is an additive process, where successivelayers of material are laid down in different shapes. After each layeris added the “ink” is polymerized, typically by photopolymerization, andthe process repeated until a 3D object is created. The recent commercialavailability and reduced cost makes 3D printing of biomolecules,including vaccines and pharmaceuticals, attractive for distribution ofthese compounds to developing countries. This would negate the need toship a finished product into the country. Instead, a 3D printer at thepoint of care can print out the required biomolecule from a simplecomputer program, which can come from anywhere in the world.

The 3D printing workflow can be described in 3 sequential steps: 1) thepowder supply system platform is lifted and the fabrication platform islowered one layer; 2) a roller spreads the polymer powder into a thinlayer; 3) a print-head prints a liquid binder that bonds the adjacentpowder particles together. Billiet et al., Biomaterials, 33:6020-6041(2012). Two kinds of 3D printing techniques are mostly adopted fornanobiomaterial fabrication. One is inkjet printing with the typicalprinters. Marizza et al., Microelectrionic Engin. 111:391-395 (2013).The other is nanoimprint lithography.

Nanoimprint lithography (NIL) is a fast and cost-efficient technique forfabricating nanostructures. The procedure of NIL is to stack multiplelayers of such structures on top of each other; that is, a finisheddouble-layer of structures is covered with a spacer-layer which isplanarized using the chemical-mechanical polishing so that a secondlayer can be processed on top. Liu et al., J. Nanomat. August. (2013).

Ink-Jet printing has been used to produce monodisperse PLGA particles.Bohmer et al., Colloids and Surfaces A: Physiochem. Eng. Aspects, 289:96-104 (2006). Briefly, droplets of a PLGA solution are printed with theink-jet nozzle submerged into an aqueous phase. This method producesmicrospheres at predictable and controllable sizes. This technique hasbeen used to created Paclitaxel-loaded monodisperse microspheres.Radulecu et al., Digital Fabrication Sep. 18-21. (2005). Variation ofthis technology has been used to create multilayer monodispersemicrospheres. See Kim and Pack, BioMEMS and Biomedical Nanotechnology,1:19-50 (2006). Utilizing this technology, microcapsule shell thicknesscan be varied from less than 2 microns to tens of microns whilemaintaining complete and well-centered core encapsulation formicrocapsules near 50 microns in overall diameter.

Drug delivery rates from microspheres have been varied by providinguniform monodisperse microparticles, mixtures of microparticles ofvarying sizes, and microparticles having different degradable layers.See Kim and Pack, BioMEMS and Biomedical Nanotechnology, 1:19-50 (2006).

Additional information can be found at internet sitestore.makerbot.com/replicator2 (2013); Tekin et al., Inkjet printing asa deposition and patterning tool for polymers and inorganic particles.Soft Matter, 4:703-713 (2008); Jang et al., Influence of fluid physicalproperties on ink-jet printability. Langmuir, 25:2629-2635 (2009); Lan,Design and Fabrication of a Modular Multi-Material 3D Printer., M. Sc.Thesis: Massachusetts Institute of Technology, 2013; and at web siteimagexpert.com/site-new/pdf/IXjetXpert.pdf. (2013).

The examples demonstrate preparation of microparticles using 3DP.Waveform was optimized for jetting monodisperse ink dropletsconsistently. The applied voltage, the duration of the applied voltage,and the change in voltage over time (slope) are all parameters that mustbe optimized to jet high quality ink drops. For example, the waveformwas optimized for a solution of 5% w/v 31 k (average molecular weight)PLGA in 1,4-dioxane. Waveforms were optimized with the JetXpert imagingsystem, and the ink and waveform and then transferred to themulti-material inkjet 3D-printer. JetXpert imaging was done with aconstant pressure waveform. In one embodiment, 5% w/v 31 kPLGA/1,4-dioxane (Z=5.3, η=6.08 mPa-s) was used. The waveform wasoptimized for this specific solution. In another embodiment, 15% w/v 12k PLGA/1,2-dichloroethane (Z=5.8, η=6.24 mPa-s) was used. Most nozzlesshowed optimal jetting as in the first embodiment, with very few nozzlesgiving sequences containing satellite drops. In a third embodiment, 15%w/v 12K PLGA/chloroform (Z=5.8, η=5.99 mPa-s) was used (Table 1). Whenusing more volatile solvents (chloroform or acetone), most nozzles firedmore than one drop continuously. In general, for a constant optimizedwaveform and similar fluid properties, inks made with more volatilesolvents led to inconsistent jetting, and print head nozzles would clogduring printing (observed when using 1,2-dichloroethane or chloroform).Inks can be made with 1,4-dioxane and DMF, to prevent nozzles fromclogging. Printing with these solvents requires longer drying timebetween layers, to prevent structures from morphing. As determined byGPC, for a given polymer, increasing the polymer concentration in theink increased the ink viscosity. For a given polymer concentration,increasing the molecular weight range of the polymers increased the inkviscosity. After polymer addition, changes observed in ink densities andsurface tensions were negligible relative to viscosity changes.

TABLE 1 Solvents for 3DP. Surface Viscos- Z number Mw Range/ DensityTension ity (dimension- Solvent % w/v (g/ml) (mN/m) (mPa-s) less)4k-15k/  5% 1.45 28.07 1.42 24.6 Chloroform 10% 1.43 27.93 3.17 10.9 15%1.44 27.67 5.03 6.9 7k-17k/  5% 1.45 27.99 2.26 15.4 Chloroform 10% 1.4127.74 3.64 9.4 15% 1.43 28.26 5.99 5.8 24k-38k/  5% 1.45 28.12 3.60 9.7Chloroform 10% 1.40 27.77 9.32 3.7 15% 1.44 28.70 26.3 1.3 4k-15k/  5%1.28 33.31 2.26 15.8 1,2-dichloroethane 10% 1.28 32.92 3.65 9.7 15% 1.2733.68 5.58 6.4 7k-17k/  5% 1.28 33.69 2.35 15.3 1,2-dichloroethane 10%1.25 32.17 3.88 9.0 15% 1.29 33.95 6.24 5.8 24k-38k/  5% 1.26 32.36 3.948.9 1,2-dichloroethane 10% 1.30 33.90 13.8 2.6 15% 1.27 33.68 49.1 0.724k-38k/  5% 1.09 31.52 6.08 5.3 1,4-dioxane 24k-38k/  5% 0.97 36.605.50 5.9 dimethylformamide

FIGS. 11A and 11B are schematics of particles made by emulsion (FIG.11A) and by 3D printing (FIG. 11B), and the release of protein from theemulsion particle (FIG. 11C) and in an ideal case the 3DP particle (FIG.11D). These show the differences in the resulting structure, which alsochanges the release kinetics.

FIGS. 12A-12D are schematics of the 3D printing process: Creating thestructure of PLGA particles (FIGS. 12A and 24A), filling drugs orproteins into the particles (FIGS. 12B and 24B), drying the drugs orproteins (FIG. 12C), and encapsulation of the particles (FIGS. 12D and24C). The schematic shows a cube shaped structure filled with vaccine,but it is understood that a mold may be utilized to provide any shaped,and that the mold may be filled in layers or more complex patterns.

Waveform parameters can be optimized to produce uniform single dropletsof “ink” (polymer solution) during printing from a piezoelectric nozzlejet (FIG. 13). Drop size is a function of various parameters which areoptimized, including applied voltage, slope of voltage, polymer andsolvent selection and concentration.

C. Micromolding

Park et al., Biomed. Microdevices, 9:223-234 (2007), describes usingmicromolding to fabricate polymer microstructures having sophisticateddesigns. Micromolds were filled with polymer microparticles, to producemicrostructures composed of multiple materials, having complexgeometries, and made using mild processing conditions. Thesemicroparticles are typically prepared using an oil-water,double-emulsion system; spray drying methods; supercritical conditioningmethods; and milling methods. In a preferred embodiment, micromolds canbe prepared by photolithographically creating a female master mold madeof photoresist, molding a male master structure out ofpolydimethylsiloxane (PDMS) from the female master mold and) molding afemale replicate mold out of PDMS from the male master structure.Polymer microparticles can be micromolded using temperature/pressmethods and/or from solvent.

Polymeric microparticles of 1 to 30 μm in size were made from PLA, PGAand PLGA using spray drying and emulsion techniques. These polymermicroparticles were filled into PDMS micromolds at room temperature andmelted or bonded together, for example, by ultrasonically weldingmicroparticles together in the mold while maintaining the voids inherentin their packing structure. Multi-layered microstructures werefabricated to have different compositions of polymers and encapsulatedcompounds located in different regions of the microstructures. Moldswere filled with solid polymer microparticles instead of a polymer meltto copy microstructures with complex geometries and composed of multiplematerials using mild processing conditions. Microparticles can floweasily into the cavities of micromolds at room temperature and lowpressure, which facilitates making microstructures with high aspectratios. Moreover, polymer microparticles can encapsulate chemicalcompounds, such as drugs, and can be filled into molds in sequentiallayers to accommodate multiple material compositions. After filling themold, the final microstructures can be created by welding themicroparticles within the mold by plastic welding methods, includingthermal and ultrasonic welding as well as solvent and gas based welding.

These same techniques can be used to formulate vaccine formulations,once has identified the polymeric materials and conditions required toobtain a narrow time of release at specific time points followingadministration.

IV. Methods of Administration

The vaccine formulations are administered to an individual in need ofvaccination. These are administered as a dosage formulation including aneffective amount of one or more antigens released in a schedule thatelicits a protective effect against the source of the antigen.

Microparticles or microcapsules can be administered by injection,preferably subcutaneously or intramuscularly, for example, under theskin of the back of the upper arm, or to a mucosal surface (orally,intranasally, via the pulmonary route, or other orifice), althoughinjection is preferred if release is to occur over a prolonged period oftime of more than a few days.

The dosage form is designed to release a bolus of antigen at the time ofadministration. This may be achieved by administering a solution ordispersion of antigen in combination with the vaccine formulationproviding multiple releases at subsequent times, or the device may beformulated to provide an initial bolus as well as subsequent release(s).

Representative vaccines are shown in Table 2:

Age Vaccine Birth 1 month 2 months 4 months 6 months 12 months 15 months18 months 19-23 months Hepatitis B¹ HepB HepB HepB Rotavirus² RV RV RV²Diphteria, DTaP DTaP DTaP See DTaP Tetanus, footnote³ Pertussis³Heamophilius Hib Hib Hid⁴ Hib influenza type b⁴ Pneumococcal⁵ PCV PCVPCV PCV Inactivated IPV IPV IPV Poliovirus⁶ Influenza⁷ Influenza(Yearly) Measles, MMR See footnote⁸ Mumps, Rubella⁸ Varicella⁹ VaricellaSee footnote⁹ Hepatitis A¹⁰ HepA (2 doses) Meningococcal¹⁰

Additional vaccines of great interest in third world countries includepolio and smallpox. The following uses polio vaccine as an exemplaryvaccine for this application.

IPV Vaccine SSI is an inactivated vaccine used for prophylacticvaccination against paralytic poliomyelitis. IPV Vaccine SSI containsinactivated poliovirus type 1, 2 and 3, propagated in Vero cells.

Contents per dose (0.5 ml):

Inactivated poliovirus type 1 (Brünhilde) 40 D-antigen units;

Inactivated poliovirus type 2 (MEF-1) 8 D-antigen units;

Inactivated poliovirus type 3 (Saukett) 32 D-antigen units;

Medium 199 to 0.5 ml.

The vaccine is manufactured without use of serum and trypsin and doesnot contain preservatives or adjuvants. Antibiotics are not used in themanufacture. IPV Vaccine SSI contains trace amounts of residualformaldehyde. It is manufactured in Denmark by Statens Serum Institut.

IPV Vaccine SSI is a solution for injection distributed in single-dosevials. For primary vaccination a series of three doses of 0.5 ml isadministered.

For booster vaccination of previously primary vaccinated persons onedose of 0.5 ml is administered, at the earliest 6 months after theprimary vaccination series. Administration of additional booster dosesshould take place in accordance with national recommendations for polioimmunization. The vaccine should be administered intramuscularly orsubcutaneously. The vaccine must not be administered intravascular. Theage at the first dose should be at least 6 weeks, and the primaryvaccination series should include at least three immunizations, with aninterval of at least four weeks. Most countries give IPV using the sameschedule as DPT vaccine (typically 2 months, 4 months, and 6 months ofage).

The immunogenicity and safety of IPV Vaccine SSI has been investigatedin several clinical trials, including clinical trials with combinedvaccines for pediatric use. Apart from IPV these trials included vaccineantigens against tetanus, diphtheria, pertussis and Haemophilusinfluenzae type b.

When initiating immunizations at two months of age, completion of aprimary vaccination series of three immunizations with at least 1 monthinterval can be expected to result in seroconversion to all three typesof poliovirus one month after the second immunization. When initiatingimmunizations before two months of age, and at the earliest at 6 weeksof age, seroconversion rates between 89% and 99% have been demonstrated.Therefore, in such a schedule, a booster dose at 9 months of age or inthe second year of life should be considered.

IPV Vaccine SSI can be used for revaccination in infants, pre-schoolaged children and adults primary immunized with IPV or OPV. IPV VaccineSSI can be used in mixed IPV/OPV schedules, using one to three doses ofIPV followed by one to three doses of OPV. It is recommended toadminister IPV before the first dose of OPV. In a mixed IPV/OPVschedule, persistence of protective antibodies after primary vaccinationhas been shown to last at least 20 years. In an IPV only schedule thepersistence of protective SSI recommended dose:

-   -   D-antigen type 1-40 DU/ml    -   D-antigen type 2-8 DU/ml    -   D-antigen type 3-32 DU/ml        10× Trivalent IPV:    -   D-antigen type 1-327 DU/ml    -   D-antigen type 2-70 DU/ml    -   D-antigen type 3-279 DU/ml

The present invention will be further understood by the followingnon-limiting examples.

EXAMPLES

The examples below describe different polymer-drug or polymer-antigenformulations for controlled release of the drug/antigen. The controlledrelease relies on polymer degradation to achieve multiple bursts ofdrug/antigen release over time following single injection.Immunogenicity of the formulations following polymer degradation-basedbursts of drug/antigen release and sustained increase in anti-antigenantibody titer are also presented. Methods of making the formulations,which include incorporation of the antigen into the polymer matrix viaspontaneous emulsion, or encapsulation of the antigen within a polymershell via 3D printing or micromolding, are also described. Studies onimproving the stability of the antigen, the stability of the polymermatrix, and the stability of the formulations, so that formulations withdesired release characteristics and immunogenicity can be obtained, arealso presented.

Example 1: Selection of Polymers and Solvents for Discrete Release ofVaccine

Materials and Methods

-   -   Polymer type—PLGA, PLLA; polymer M_(w)—9.5 k, 20 k, 31 k, 46 k    -   Drug loading—0.5, 3, 5%; excipients—trehalose, sucrose    -   Encapsulation—Spontaneous Emulsion        The process used CH₂Cl₂:TFE::4:1. (CH₂Cl₂—dichloromethane        TFE—trifluoroethanol) as the organic phase, with poly(vinyl        alcohol) (“PVA”) to encapsulate 5%, 3% or 0.5% bovine serum        albumin (“BSA”) or inactivated polio virus (“IPV”) into the        polymer. The addition of the organic phase to a large volume of        a non-solvent phase forms a spontaneous single emulsion and the        resulting solution is stirred continuously to evaporate the        solvent. Immediate formation of microspheres occurs. After        stirring, the microspheres are washed and then dried. See        Jaklenec et al., Sequential release of bioactive IGF-I and        TGF-β1 from PLGA microsphere-based scaffolds. Biomaterials,        29(10):1518-1525 (April 2008).

Release studies were done in vitro with 10 mg of microspheres suspendedin 1 ml of PBS buffer (pH 7.2) at 37° C. Time points where taken at day1, 4, 7 and every week thereafter. At each time point, the vials werecentrifuged, the supernatant was removed to be assayed, a fresh 1 ml ofPBS was added and the pelleted microspheres were resuspended.

Results

FIG. 21A is a graph of the release of a model protein, bovine serumalbumin (“BSA”) over time (weeks) for 5% BSA, 3% BSA, and 0.5% BSA fromPLGA (50:50), 20 kD, derivatized with a carboxylic group.

FIG. 21D is a graph of the release of bovine serum albumin over time(weeks) for 5% BSA, 3% BSA, and 0.5% BSA from PLGA (50:50), 9.5 kD,derivatized with a carboxylic group.

These figures show release from the same polymer, but with differentmolecular weights. This difference alone dramatically changed releaseprofiles.

FIGS. 3A and 3B are graphs of the release of bovine serum albumin overtime (weeks) for 5% BSA and 0.5% BSA from PLLA, 50 kD.

FIGS. 4A and 4B are graphs of the release of bovine serum albumin overtime (weeks) for 5% BSA and 0.5% BSA from PLLA, 100 kD.

FIGS. 5A and 5B are graphs of the release of bovine serum albumin overtime (weeks) for 5% BSA and 0.5% BSA from PLLA, 300 kD.

The only difference between these three graphs is the molecular weight.However, in no case were there substantial, distinct periods of releasesimilar in scope to those in FIG. 21.

FIGS. 6A and 6B are graphs of the release of bovine serum albumin overtime (weeks) for 5% BSA and 0.5% BSA from P(d,l)LA, 20 kD. The resultsare similar to FIGS. 3-5 and 21.

The best results were obtained using PLGA-COOH (50:50). This issummarized in FIGS. 7A and 7B for PLGA formulations (FIG. 7A) comparedto bolus injections (FIG. 7B).

The details of the formulations used for FIG. 7A are presented in Table3 (see also Tables 4 and 5). In Table 3, the difference between F3 andF7 is the Mw of the polymer. F5 has high loading of BSA. Note that F3and F7 have about 3 equivalent doses, while F5 has a large initial dosefollowed by two smaller ones—this is very similar to an initial boluswith 2 booster shots. Formulations are injected into animals on day 0(single injection), while bolus controls are injected 3 times during thecourse of the study to mimic formulation release kinetics. Negativecontrols include blank microspheres for formulations and saline forbolus injections. Injection volumes were 200 μl (max volume forsubcutaneous (SC) injection in mice). Two injections of 200 μlinjection, one per leg, were performed. Maximum injectable microsphereconcentration was 50 mg/ml.

TABLE 3 Amount of BSA released (μg) at first, second and third peaksfollowing injection of F3, F5 and F7 formulations. Amount of BSA (μg)1^(st) 2^(nd) 3^(rd) Groups (n = 10) peak peak peak Total F3: 0.5% BSA,20k BI-502H, 22.2 19.8 21.6 63.6 50/50 F7: 0.5% BSA, 31k PLGA, 50/5023.0 13.6 34.0 70.6 Bolus control (2) 22.0 22.0 22.0 66.0 F5: 5% BSA,31k PLGA, 50/50 298.0 68.4 65.4 431.8 Bolus control (1) 298.0 68.4 65.4431.8

FIGS. 8A and 8B are graphs of the release of 0.5% of bovine serumalbumin compared to release of 0.5% of ovalbumin over time (weeks) fromPLGA (50:50), 20 kD, derivatized with a carboxylic group.

FIGS. 9A and 9B are graphs of the release of 5% of bovine serum albumincompared to release of 5% of ovalbumin over time (weeks) from PLGA(50:50), 31 kD, derivatized with a carboxylic group.

FIGS. 10A and 10B are graphs of the release of 0.5% of bovine serumalbumin compared to release of 0.5% of ovalbumin over time (weeks) fromPLGA (50:50), 31 kD, derivatized with a carboxylic group.

These all shows excellent results, varying only due to the molecularweights of the polymers or the percent loading.

Example 2: Predicted Release Profiles from Microparticles Made byEmulsion Compared to Microparticles Made by 3DP

Particles fabricated by computer-controlled inkjet 3D-printing can bemade with identical micrometer-scale dimensions, drug loadings, andspatial locations of drugs within the polymer microstructures. The maindifference between the two methods is that emulsion based particles(left) are matrix based and the drug is homogeneously distributedthroughout the particle. The 3D or micromolded particle is shown on theright. The vaccine and polymer are distinctly separated, where thevaccine is in the core and the polymer is only in the shell. Thesedistinctions allow for unique control of release kinetics between thetwo particle types. Also, the micromolded/3D printed particle allows forhigher loadings since the core size can also be controlled.

The release graph of FIG. 11C is based on the data in Example 1.

In the schematic of FIG. 11E, the drug or the vaccine is encapsulated inpolymer microspheres and dispersed throughout the polymer matrix (A).Once hydrated, any drug on the surface is immediately released (B) thuscausing the initial burst. Following this event, the microspheres haveremnant pores through which drug can slowly diffuse causing thesecondary burst (C). Finally, when the polymer bulk erodes, due tosignificant Mw degradation, the reminder of the drug is released in athird burst (D).

Example 3: Selection of Parameters for Piezoelectric Jetting to MakeMicroparticles

Materials and Methods

Optimization:

Correct the applied pressures from the nozzles (waveform) to jet outuniform, single droplets of PLGA

Optimize viscosity of the PLGA solution (Z number)

Low enough viscosity to jet out efficiently from the nozzle

High enough viscosity to solidify on the substrate

Studies have shown that proper drop formation in piezoelectricdrop-on-demand (DOD) ink-jet printing can be described using thedimensionless Z number. The Z number is determined by the ink's fluidproperties (surface tension, density, and viscosity) and the size of aprinthead's nozzle.

Z number ranges can be defined for determining ink printability with aspecific waveform.

Characterize PLGA solutions using the Z number:

Solvents are chosen based on PLGA solubility and physical properties(i.e. vapor pressure and boiling point).

Once an optimal waveform is determined for one PLGA solution ink, thatsame waveform can be used to print with different MW PLGAs, by usingPLGA solutions that have Z numbers within a certain range.

Results

The waveform for jetting BSA and IPV solutions was optimized as shown inFIG. 13. This allows filling of the polymer printer particles withvaccine.

Example 4: Studies to Minimize Loss of IPV D-Antigen and IncreaseStability During Lyophilization and Encapsulation

Studies were conducted to optimize methods and reagents forconcentrating IPV prior to encapsulation, to prevent loss of D-antigendue to lyophilization. Methods that can be used include centrifugalfilters and dialysis.

Excipients that may reduce D-antigen loss during emulsion process weretested. BSA has been used with some success. The current studiesutilized sugars. Varying sugar concentrations for forming sugar glasswas tested. Varying humidity conditions for drying was also tested.

Comparisons with other solvent systems for the spontaneous emulsionprocess were also made. In the initial studies, chloroform/acetone wasused for the organic phase. In subsequent studies, ethyl acetate wasused as a substitute for dichloromethane (DCM).

Materials and Methods

Alcock et al. reported that viruses incorporated into sugar glassdisplay long-term solid-state stability. (Alcock et al., ScienceTranslational Medicine, 2(19):19-19ra12 (2010)) IPV stability in3D-printed substrates was tested in a similar manner. IPV solutionscontaining co-dissolved sugars (sucrose and trehalose) were depositedonto a scaled-up PLA structure printed using the MakerBot Replicator 2.Drying of the IPV/sugar solutions was done at ambient humidity (˜20%humidity), which was higher than that used by Alcock et al. (˜10%).

Results

FIGS. 14A, 14B and 14C are graphs of the percent D-antigen (IPV type I,II, and III) retained after lyophilizing with sugar excipients 1 Mtrehalose, 1 M sucrose, and 3 M sucrose, then incubating at 4° C., 25°C. or 37° C.

The results demonstrate that the sugar significantly increased thestability of the IPV, and that there were few differences betweenstability at 4° C. and 25° C., while stability was less at 37° C. Theresults also showed that the stability is different depending on the IPVtype.

FIGS. 15A and 15B are graphs of the percent D-antigen (IPV type I, II,and III) retained after lyophilizing with 0, 0.5, 0.75, 1 or 1.25 Mtrehalose, 1 then incubating at 4° C. (FIG. 15A) or 25° C. (FIG. 15B).The best results were obtained with 0.5 M and 0.75 M trehalose.

FIGS. 16A and 16B are graphs of the percent D-antigen (IPV type I, II,and III) retained after mixing with solvents until solvent evaporation,with solvents tetrafluoroethylene (“TFE”), dichloromethane (“DCM”), orTFE and DCM, without sugar excipient (FIG. 16A) or with 0.75 M trehaloseas excipient (FIG. 16B). DCM was statistically significantly better. IPVwas not stable when exposed to the organic solvents during theencapsulation process, even when trehalose was used as an excipient.

FIG. 17A is a graph of the percent D-antigen (IPV type I, II, and III)retained after IVP with 0 M sugar, 0.5 M trehalose, or 0.5 Mtrehalose-sucrose is printed onto 3D-printed PLA substrate and dried at25° C., 22% RH. FIG. 17B is a graph of the percent D-antigen (IPV typeI, II, and III) retained after IVP with 0.25 M trehalose or 0.5 Mtrehalose-sucrose is printed onto 3D-printed PLA substrate and dried at25° C., 10.7% RH.

Alcock et al. allowed virus/sugar solution to dry onto substrate undercontrolled conditions (20-25° C., 2-10% RH). Drying for FIG. 17Ainvolved IPV/sugar solutions on PLA substrates within a ventilated cellculture hood (20-25° C., 22.9% RH). Drying for FIG. 17B involvedIPV/sugar solutions on PLA substrates in a desiccator (20-25° C., 10.7%RH). Stability was better for room temperature/humidity (all three IPVtypes had >50% D-antigen retained).

FIGS. 18A and 18B are graphs of the percent D-antigen retained afterlyophilization with 0.5 or 0.75 M trehalose then incubating with 25° C.(FIG. 18A) or after lyophilization with 0.5 M trehalose-sucrose,pipetted, or 0.5 M trehalose-sucrose, jetted, then drying at 25° C.,10.7% RH (FIG. 18B).

These studies demonstrate that the best results were obtained by storingthe 3D-printed PLA particles containing lyophilized IPV at roomtemperature and humidity (˜20% relative humidity) The studies alsodemonstrated that jetting the IPV, rather than lyophilizing the IPV, didnot significantly decrease IPV stability.

Example 5: Short-Term In Vivo Immunogenicity of BSA-Containing PLGAFormulations

The immune response, presented as the anti-BSA IgG (antibody) titer, wasmeasured at 1, 2, and 4 weeks following injection of formulations F3, F5and F7, presented in Table 3 above and Table 4 below. Formulations F4and F8 are blank microspheres (no drug), used as negative controls (seeTable 4).

The results are presented in FIGS. 19A, 19B, and 19C. The antibody titer(log 2) is plotted for the various groups at 1, 2, and 4 weeks. Thenegative controls are all zero and the microsphere formulations aregenerating an equivalent or stronger response compared to the boluscontrol.

Example 6: Long-Term In Vivo Immunogenicity of BSA-Containing PLGAMicrospheres

Materials and Methods

Materials

Poly(D,L-lactic-co-glycolic acid) (PLGA Resomer® RG 502 H, RG 503 H, RG504 H, and RG 752 H) and BSA were purchased from Sigma-Aldrich (St.Louis, Mo.). Poly(vinyl alcohol) (PVA, Mw=25,000) was purchased fromPolysciences, Inc. (Warrington, Pa.). Dichloromethane (DCM) and2,2,2-trifluoroethanol (ME) used in this study were reagent grade.

Microsphere Fabrication

Sixteen formulations of PLGA microspheres containing BSA (Table 4) werefabricated using a spontaneous single-emulsion method (Fu et al., JPharm Sci, 92:1582-1591, 2003; and Jaklenec et al., Biomaterials,29:185-192, 2008). Briefly, 200 mg of PLGA were dissolved in 10 mL of4:1 DCM:TFE and mixed with 300 μL of BSA in water. Mixing formed aclear, single-phase solution that was then added to 200 mL of 5% (w/v)PVA in water. The emulsion formed spontaneously and was stirred at roomtemperature for 3 hours. Particles were then collected viacentrifugation, washed five times with water, and lyophilized. Whenprepared for in vivo use, organic phase and BSA solutions were filteredthrough 0.2 μm polytetrafluoroethylene filters (Whatman, LittleChalfont, England) and combined in a sterile laminar flow hood.

Microsphere Characterization

Microsphere size distribution was determined using a Multisizer 3Coulter Counter. Histograms were created using a bin size of 0.39 μm andsmoothed using central moving average with a window size of ±5 bins.Scanning electron microscope (SEM) images were collected using aJSM-5600LV SEM (JEOL, Tokyo, Japan) at an acceleration voltage of 5 kV.Prior to imaging, samples were coated with Au/Pd using a Hummer 6.2Sputtering System (Anatech, Battle Creek, Mich.) to prevent surfacecharging.

TABLE 4 Microsphere formulations and size characterization. Particle 90%of BSA PLGA M_(w) PLGA Size Particles Formulation (% w/w) (kDa) Ratio(μm) Below (μm) A (F1) 5  7-17 50:50 10.5 ± 6.8 18.5 B (F2) 3  7-1750:50 10.6 ± 6.4 18.1 C (F3) 0.5  7-17 50:50 10.3 ± 6.2 22.1 D (F4) 0 7-17 50:50 10.5 ± 5.9 17.4 E (F5) 5 24-38 50:50  8.6 ± 6.7 21.4 F (F6)3 24-38 50:50 11.4 ± 8.3 21.4 G (F7) 0.5 24-38 50:50 12.1 ± 8.2 23.1 H(F8) 0 24-38 50:50 11.4 ± 7.2 20.2 I (F9) 5 38-54 50:50 14.1 ± 9.4 25.4J (F10) 3 38-54 50:50 12.0 ± 7.1 20.3 K (F11) 0.5 38-54 50:50 11.9 ± 6.820.6 L (F12) 0 38-54 50:50 11.9 ± 6.4 19.7 M (F13) 5  4-15 75:25 11.3 ±7.0 19.7 N (F14) 3  4-15 75:25 12.2 ± 7.4 21.2 O (F15) 0.5  4-15 75:2512.4 ± 7.5 21.1 P (F16) 0  4-15 75:25 11.7 ± 6.5 19.9

In Vitro BSA Release

Ten milligrams of microspheres were dispersed into 1 mLphosphate-buffered saline (PBS) in capped tubes and incubated on arotating platform at 37° C. At each time point (1 day, then weekly for1-13 weeks), samples were centrifuged at 1500 relative centrifugal force(RCF) for 5 min, after which the supernatant was collected. Samples werethen resuspended in fresh PBS and returned to the incubator for samplingat subsequent time points. BSA release from microspheres was quantifiedusing a bicinchoninic acid (BCA) assay and normalized to the totalamount released by the end of the study. Samples were run in triplicateand data reported as mean±standard deviation.

In Vivo Administration of BSA Microspheres

All animal work was approved by MIT's Committee on Animal Care. Briefly,female BALB/c mice between 6 and 8 weeks of age received injections of(1) BSA-loaded microspheres, (2) unloaded microspheres, (3) bolus BSA,or (4) saline-only. While mice in the first two groups received only oneinjection, those receiving a bolus BSA or saline-only were injectedagain at 4 and 8 weeks to match the amount and timing of BSA releasefrom PLGA microspheres in vitro. Samples were dissolved or suspended(when applicable) in 200 μl of saline and injected subcutaneously intoeach hind limb for a total of 400 μl. Table 5 contains the exact dosingregimen for each group. At week 0, 1, 2, 4, 6, 8, and 10, 100 μl ofblood was sampled sub-mandibularly and, after clotting, was centrifugedat 2000 RCF for 10 minutes at 4° C. to separate the serum.

Immunogenicity of BSA Release from PLGA Microspheres

Serum antibody titers against BSA were determined using an endpointenzyme-linked immunosorbent assay (ELISA). 96-well Maxisorp ELISA plates(Thermo Fisher Scientific, Waltham, Mass.) were coated overnight at 4°C. with 100 μL of a 100 μg/ml solution of BSA in 0.1M sodiumbicarbonate. Plates were then washed three times in PBS containing 0.05%Tween 20 (PBST) and then incubated in 5% non-fat milk in PBST for 2hours at 37° C. as a blocking agent. Following another series of threewashes with PBST, mouse serum samples were added in four-fold serialdilutions and incubated for 2 hours at 37° C. The extent of serumdilution increased as the study progressed and titers rose. Plates werethen washed live times with PBST and incubated at 37° C. withhorseradish peroxidase-conjugated goat anti-mouse secondary antibody(Southern Biotechnology Associates, Birmingham, Ala.) diluted 1:1000 inblocking buffer for 2 hours. Plates were washed an additional five timeswith PBST and developed using 100 μL of p-nitrophenyl phosphate solutionprepared from tablets dissolved in 1× diethanolamine buffer from analkaline phosphate substrate kit (Bio-Rad, Hercules, Calif.). After 10minutes, the reaction was stopped by adding 100 of 0.4M sodium hydroxideto each well and absorbance values were read at 405 nm using a TecanInfinite M200 Pro microplate reader (Männedorf, Switzerland). Titerswere reported as the reciprocal of the highest serum dilution thatyielded an absorbance greater than 2-fold above background values.

TABLE 5 Dosing regimen for in vivo BSA administration. Amount of BSA(μg) First Second Third Group Dose Dose Dose Total Formulation C (0.5%BSA, 7-17 22 20 22 64 kDa PLGA)* Formulation G (0.5% BSA, 24- 23 14 3471 38 kDa PLGA)* Low Dose Bolus BSA 22 22 22 66 Formulation E (5% BSA,24-38 298 68 65 431 kDa PLGA)* High Dose Bolus BSA 298 68 65 431Unloaded PLGA microspheres, 0 0 0 0 7-17 kDa Unloaded PLGA microspheres,0 0 0 0 24-38 kDa Saline 0 0 0 0 All PLGA used in vivo was 50:50.*Indicates theoretical dose based on in vitro results.

Statistical Analysis

All data in Table 4 and FIG. 20 are reported as mean±standard deviation.In vitro studies were performed in experimental triplicate while in vivostudies were performed with ten experimental replicates for all groupsat all time points, except Formulation C at week 10 in which ninereplicates were used due to insufficient blood volume from one animal.Time-matched antibody titers were compared using one-way ANOVAstatistical analysis for the three low dose groups and Student'sunpaired two-tailed t-test for comparing the high dose microspheres andbolus injections. Comparisons of peak antibody titers were performedusing Student's unpaired two-tailed t-test with the Holm-Bonferronicorrection at a significance level of 0.05 to counteract the effect ofmultiple comparisons.

Results

Characterization of BSA-Containing PLGA Microspheres

All sixteen formulations of PLGA and BSA produced sphericalmicroparticles with broad distributions of particle sizes (Table 4). Thesize and shape of Formulations C, G, and E, which were used for in vivostudies, were representative of all formulations. Formulation C producedmicrospheres that were 10.3±6.2 μm in diameter yet because of the cubicrelationship between volume and diameter, particles smaller than 10.3 μmcontained just 4.2% of the antigen load assuming homogeneousdistribution of BSA in PLGA (Narasimhan et al., J Control Release,47:13-20, 1997). Larger particles contained a majority of the antigenwith 90% of the volume contained in particles larger than 22.1 μm forFormulation C. Formulations G and E demonstrated similar characteristicswith particle diameters of 12.1±8.2 and 8.6±6.7 μm respectively, yetwith 90% of particle volume contained in particles larger than 23.1 and21.4 μm respectively. Histograms of microsphere diameter and volumedistribution can be seen in FIG. 20A-20F. Particles at the large end ofthe distribution also contributed substantially to surface area effectsas 50% of the cumulative particle surface area was present on particleslarger than 23.67, 29.54, and 27.58 μm in diameter for Formulations C,G, and E respectively.

In Vitro Release of BSA from PLGA Microspheres

In vitro release kinetics from PLGA microspheres were determined by BCAassay and expressed as a percentage of the total BSA released during theduration of the experiment. BSA release and particle degradationoccurred more quickly in PLGA microspheres fabricated using any of thethree polymers with 50:50 ratios of lactic-to-glycolic acid compared toa 75:25 ratio. All microsphere formulations produced using PLGA with a50:50 ratio degraded within 14 weeks whereas 75:25 PLGA degraded in 22weeks. The timing of BSA release appeared to be more dependent onpolymer type than BSA loading, though loading had a major effect on thesize of the bursts in most cases. Low molecular weight (7-17 kDa) PLGAreleased in three distinct bursts over the course of 8 weeks andcompletely degraded by week 14 as seen in FIG. 21A. The medium molecularweight (24-38 kDa) PLGA also displayed three bursts spread over 9-12weeks depending on BSA loading and degraded by week 14 (FIG. 21B). Thehighest molecular weight (38-54 kDa) PLGA released BSA over 9-12 weekswith prominent bursts at day 1 and week 8, but with more continuousrelease kinetics in between from the 3 and 5% BSA loaded microspheres(FIG. 21C). Microspheres made from low molecular weight (4-15 kDa) PLGAwith a higher lactic acid content (75:25) degraded over 22 weeks andshowed gradual semi-continuous release after an initial burst (FIG.21D).

Out of the sixteen in vitro formulations, Formulations C, G, and E werechosen for the subsequent in vivo study based on their in vitro releasekinetics. Formulation C microspheres were made using low molecularweight PLGA (7-17 kDa) loaded with 0.5% BSA, Formulation G with slightlyhigher molecular weight PLGA (24-38 kDa) and 0.5% BSA, and Formulation Ewith the same molecular weight PLGA as Formulation G, but with higher(5%) BSA loading. BSA release from Formulation C was characterized bythree distinct peaks at day 1 when 33.4±5.1% of total BSA was released,at week 4 when 27.2±4.3% was released, and across the week 6 and week 7time points during which 32.1±5.2% was released respectively (FIG. 21A).Minimal BSA release was observed at weeks 1, 2, 4, and 5, or after week7 and microspheres were completely degraded by week 10 as evidenced bycomplete dissolution of the particles.

Formulation G microspheres were also characterized by BSA release inthree bursts (FIG. 21B), but spread out over a longer timeframe. Inaddition, total microsphere degradation was observed after 14 weeksrather than 10 weeks for Formulation C. The first BSA burst fromFormulation G was observed at the 1-day time-point, releasing 28.0±5.7%of total BSA. This was followed by a second burst of 12.9±2.9% at week4, and an elongated burst at weeks 8 through 11 during which acumulative 43.8±2.0% of BSA was released. Little-to-no BSA was observedin the release media at any other time points through completedegradation of the particles.

Formulation E microspheres released 63.7±7.3% of its BSA at day 1 whichwas the largest initial burst in terms of both total quantity andpercentage of any of the formulations (FIG. 21C). The only time pointswith substantial BSA release following this initial burst were weeks 3and 8 when 8.9±0.9% and 8.8±2.1% of the total load was releasedrespectively. Similar to Formulation G, which used PLGA at the samemolecular weight (24-38 kDa), Formulation E degraded completely at 14weeks.

Immunogenicity of BSA-Containing PLGA Microspheres

The humoral immune response to each microsphere formulation was comparedwith a positive control consisting of three bolus injections matchingthe quantity and timing of BSA released from particles in vitro. Withinexperimental groups, a statistically significant increase in titerbetween consecutive weeks was used as evidence of release (one-samplepaired t-test). Formulation C induced a significant increase in antibodytiter compared to the previous time point at weeks 1, 2, and 4 (p<0.05,p<0.0001, and p<0.001 respectively), then decreased significantly atweeks 6 and 8 (p<0.01 and p<0.05 respectively), before stabilizing atweek 10 (FIG. 22). Similarly, mice receiving Formulation G showed asignificant increase in titer at weeks 1, 2, and 4 (p<0.05, p<0.001, andp<0.01 respectively), remained steady at week 6, and then fellsignificantly by week 8 (p<0.05) before stabilizing again at week 10.Formulation E induced a similar response as antibody titers increasedsignificantly at weeks 1, 2, and 4 (p<0.001 for all), leveled off atweek 6, and then decreased through the end of the study (p<0.05).Overall, the immune response to all three microparticle formulationsdemonstrated a similar progression over time as titers rose over thefirst 4 weeks then slowly decreased through week 10 (FIG. 22). However,the magnitude of antibody titers appeared highly dependent on BSAloading. Based on in vitro experiments, Formulation E releasedapproximately 13 times more BSA than Formulations C at the earliest timepoint (1 day) due to a large initial burst and induced antibody titersthat was 13-fold higher as well. This trend was also observed at the endof the study as the antibody titer induced by Formulation E was 8-foldhigher than those associated with Formulation C after releasing 7 timesas much BSA.

Antibody titers from animals receiving any of the BSA-loaded microsphereformulations were significantly higher than those from animals receivingdose-matched bolus injections after 2 and 4 weeks (p<0.05 and p<0.01respectively). However, after boosting with a second bolus injection atweek 4, titers were statistically similar between formulations and theirdose-matched bolus group at 6 and 8 weeks. Then at week 10, followingadministration of the third bolus, animals receiving bolus BSA showedanother spike in titer resulting in significantly higher antibody titerscompared to all dose-matched microsphere groups at that time point(p<0.001).

While time-matched antibody titers suggest the timing of antigenpresentation, peak antibody titers may be more useful indicators ofimmune response in this case due to a possible mismatch in the timing ofantigen presentation (Paryani et al., J. Pediatr, 105:200-205 (1984);Barraclough et al., Am J Kidney Dis, 54:95-103 (2009)). The bolusinjection schedule was chosen to match the timing of microsphere burstsin vitro, but accelerated in vivo degradation could have resulted in anoffset in antigen release. As a result, at the end of the experimentantibody titers in the microsphere groups had been falling for weeks aswould be expected in the absence of antigen, whereas titers in the boluscontrol groups reached their highest levels following the thirdinjection. Comparing peak antibody titers helps to control for theeffects of timing and determine which treatment was more immunogenic.

Antibody titers for Formulations C, G, and E peaked at 13.9±1.3,13.7±2.2, and 16.1±2.1 on a log 2 scale respectively 4 weeks aftermicrosphere administration, whereas the small and large dose-matchedboluses peaked after 10 weeks at 15.5±1.5 and 17.7±0.8 log 2 titerrespectively (FIG. 23). Formulations C and G (FIG. 23A) induced peakantibody titers that were not statistically different (p*=0.0645 andp*=0.0543 respectively) than the dose-matched bolus control consistingof three 22 μg BSA injections, using the Holm-Bonferroni correctionmethod that has been recommended for multi-group titer comparisons(Reverberi, Blood Transfus. 6:37-45 (2008)). Formulation E (FIG. 23B)also induced peak titers that were not statistically different(p*=0.0784) than the dose-matched bolus control consisting of threebolus injections (Table 5).

Example 7: PLGA Micromolded Particles for Drug Delivery

Materials and Methods

Development of single drug particles involves three major steps: 1)shell microfabrication, 2) vaccine/drug filling and 3) particle sealing.

The shell microfabrication may use mask fabrication (lithography),fluoro-mold fabrication (UV core) and heat pressing of PLGA (120° C. for30 min). The step of drug filling may be carried out by the Biodotrobot, filling the core with 1.5 nL volume of the drug. The sealing isachieved by placing a PLGA cap on the shell and then sealing theparticles at 37° C. for 5 min and acetone vapor for 5 min.

The schematic of the process is presented in FIGS. 12A-12D and 24A-24C.These microparticles may be manufactured as stacks, sealing each onewith another at 37° C. for 5 min.

Results

The micromolded particle shells have an x, y, and z dimensions of450×450×300 μm with a core of 100×100×100 μm (cap dimensions are450'450×150 μm), or 200×200×150 μm, with a core of 100×100×100 μm. Thesedimensions allow the particles to be delivered with a 21 gauge needle(inner diameter 514 μm) or with a 23 gauge needle (inner diameter 337μm).

Example 8. Single PLA Particles for Depositing Drug/Sugar Solution

Materials and Methods

IPV in 0.5 M trehalose and 0.5 M sucrose solution was deposited onto PLAparticles then dried in the stem cell culture hood at room temperature.PLA particles may be 3D-printed using a Makerbot printer.

Results

Single PLA particles may serve as a useful platform for drying onto anddelivering drugs and antigens, as this method allows to deposit drug orantigen in a sugar solution onto a substrate such that the drug orantigen is stabilized within a sugar glass. Studies on the stability ofIPV antigens on PLA particles are presented in Examples below.

Example 9: Stability of the IPV Microspheres

The process of IPV microsphere formation presented in Example 1 aboveholds challenges for IPV stability during the manufacturing process.These challenges with a summary of the findings that overcome thechallenges are presented in Table 6.

TABLE 6 Summary of obstacles to IPV stability, approaches taken toovercome the obstacles and findings. Obstacle Approach Findings IPV isnot stable Double emulsion method keeps IPV in an Double emulsioniseffective in organic solvents aqueous “bubble.” Minimal direct Vortexingand sonication or at organic/aqueous interface contact with organicsparameters can be tuned to Co-encapsulate IPV with BSA or mild maximizeIPV recovery surfactant to reduce IPV exposure to Excipients improverecovery aqueous/organic interface during Gelatin emulsification Tween(polysorbate) 80 IPV may not survive the physical Use lower-energymixing: homogenization, mixing processes that form the vortexing,low-amplitude sonication, etc. emulsion IPV is not stable after dryingChange drying method (lyophilization, air- Microspheres dried at roomdrying, vacuum) temp for 1 hr undervacuum show high IPV recovery Addexcipients during drying Sorbitol/MSG/MgCl₂ improves recovery duringdrying Co-encapsulate with excipients Sorbitol/MSG/MgCl₂ can beco-encapsulated in w/o/o microspheres IPV is not stable in microsphereCo-encapsulate IPV with excipients to help Gelatin and matrix inhydrated, 37 C. maintain stability sorbitol/MSG/MgCl₂ improveenvironment stability overtime IPV is not stable in polymerCo-encapsulate IPV with basic excipients IPV is not stable <pH 6 or >pHdegradation products to buffer acidic byproducts 8 Mg(OH)₂ buffersacidic byproducts Microspheres do not show bursts of Change polymercomposition (PLGA MW, PLGAs of different MW and release at the desiredtime points ratio of LA:GA) ratios are being tested with BSAmicrospheres Change excipients added to polymer Microspheres containingpH- affecting additives are being tested

Example 10: Co-Encapsulation of IPV with Gelatin or Mild Surfactant andSonication Increase IPV Stability During Emulsification

Materials and Methods

IPV was mixed with water and/or excipients at 100:1 w/w ratio.Excipients used were gelatin, maltodextrin, pullulan, myristic acid, andTween80. IPV with excipients was then added to DCM, or PLGA/DCM. Themixture was vortexed for 10 sec on highest setting, or was sonicated 10sec at 25% amplitude. ELISA buffer (1% BSA, 1% Triton-X 100 in PBS) wasadded, the mixture was vortexed for 10 sec on high setting, and layerswere separated by centrifugation. The aqueous layer was removed andtested by ELISA.

Results

Gelatin and Tween80 both improved the stability of type I, II and IIIIPV (FIG. 25).

Type I and III IPV, generally the most sensitive to damage, showed bestrecovery with sonication at 20% amplitude for 30 sec (FIG. 26).

Example 11: Drying of IPV and Excipients with Genevac Improves IPVRecovery

Materials and Methods

IPV was concentrated 26× and mixed with pullulan, pullulan/BSA, orsorbitol/MSG/MgCl2 at 100:1, 350:1, or 700:1 mass ratio. IPV withexcipient was dried by lyophilization (overnight) or using Genevac (1hr, 30-35° C.). Recovery was tested by ELISA after resuspending thedried IPV with excipients.

Results

In all paired groups of lyophylization (Lyo) and Genevac (FIG. 27),Genevac showed higher recovery than lyophilization.

Example 12: Lyophilized IPV Shows Long-Term Stability at 37° C.

Materials and Methods

IPV was mixed with excipients 10% sorbitol, 8.5% MSG, and 8.5% MgCl₂ at40,000:1, and lyophilized in plastic tube. The lyophilized IPV withexcipients was stored in a plastic tube in a pouch with desiccant athumidified atmosphere and 37° C. for up to 30 days. At various timepoints, the lyophilized IPV was resuspended and tested for recovery byELISA.

Results

Types I, II and III IPV in lyophilized form with excipients showedlong-term stability when stored at 37° C. (FIG. 2).

Example 13: Changes in pH of Release Medium by PLGA Particles

PLGA degrades by bulk erosion leading to acid buildup insidemicrospheres. During in vitro storage, finite volume of buffer may causeacid build up in tubes, which may lead to particle erosion and acidbuild up inside the microspheres. In in vivo applications, the buffer inlocal environment is constantly replenished with minimal acid build upoutside of the particles. Because PLGA degrades by bulk erosion, acidbuild up inside the microspheres may be a problem. Therefore, severalPLGA molecules were tested for changes in the pH of the release mediumover time, when the buffer was changed every 2-3 days, or every 7 days.

Results

Results in FIG. 29 demonstrate that PLGA molecules alone change the pHof the release medium making it more acidic over time. Although changeof buffer every 2-3 days helps ameliorates this effect, it does notsolve the problem.

Example 14. Excipients to Buffer the Acidic Products of PLGAMicrospheres

It was observed that Type I IPV was relatively stable at pH 7.4 at 37°C., and type II and type III IPV were stable at pH 6-8 at 37° C.Therefore, several excipients were tested for their buffering capacityto buffer the acidic products of PLGA microspheres.

Materials and Methods

Buffering agents, such as Mg(OH)₂, Al(OH)₃, and myristic acid, wereincorporated into PLGA microspheres to minimize changes in pH of releasemedium. Also, Mg(OH)₂ has little to no solubility in water, it remainsdistributed throughout the polymer matrix and can potentially buffer theinternal compartment. Al(OH)₃ is a known adjuvant and can increaseimmunogenicity

Results

The results in FIG. 28 demonstrate that Mg(OH)₂ was able to bufferacidic byproducts in the release medium and stabilize the pH of therelease medium at about pH 7.

Example 15. IPV Microspheres Incorporating Stabilizing Excipients

Based on the investigations of Examples 9-14, PLGA microspheresincorporating IPV and various stabilizing additives were generated.These formulations are presented in Table 7 below, and were made withPLGA 502H. Other formulations with higher MW polymers, like PLGA 503H,are also contemplated.

Example 16. IPV Stability after Drying on PLA Allows Use of LowerExcipient:Vaccine Ratios

D-antigen retention after drying 26× Trivalent IPV on PLA for 16 hoursat room temperature and humidity was tested. Excipient used was 10%sorbitol, 8.5% MSG, 8.5% MgCl₂. The ratios of excipient to vaccinetested were 300 to 1, 100 to 1, 60 to 1, 30 to 1, or no excipient. Thepercent of D-antigen retained was assayed by ELISA.

Results

Upon drying of the solutions on PLA, no significant drop in stabilitywas observed when going down to a 30:1 ratio (FIG. 1). This dataindicated that concentrated excipient-free IPV could be dried on PLAcubes without much loss of antigen activity.

Example 17. Other Formulations of Concentrated IPV Mixed with Excipientsand Dried in a PLA Cube

The formulations listed in Table 8 represent % D-antigen retained whenconcentrated IPV was mixed with indicated excipient solutions (columns“Polyol” and “Other”) at the indicated ratios (column “Excip:Vax,” allindicated ratios are 100:1) and dried in PLA cubes overnight.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

TABLE 7 IPV microsphere formulations incorporting stabilizing additivesfor polymer degradation-based bursts of drug/antigen release. S/M/Mrefers to 10% sorbitol, 8.5% MSG, 8.5% MgCl_(2.) mg Approx. DUIPVpolymer loaded (Type I/ Additives to Additives to PLGA Outer stirringphase Mixing (1^(st) Mixing (MW) Type II/Type III IPV phase phase(2^(nd) emulsion) emulsion) speed/time Drying 20 216/62/187 4 mg gelatinNone Oil: Sonicator 20% amplitude 22 mg S/M/M (12k) mineral oil + Span80 30 sec 20 216/62/187 4 mg gelatin 10% myristic acid Aqueous:Sonicator 20% amplitude 22 mg S/M/M (12k) 1% PVA, 5% sucrose 30 sec 20216/62/187 4 mg gelatin 10% Al(OH)₃ Aqueous: Sonicator 20% amplitude 22mg S/M/M (12k) 1% PVA, 5% sucrose 30 sec 20 216/62/187 8.65 mg None Oil:Sonicator 20% amplitude No excipient (12k) S/M/M mineral oil + Span 8030 sec 20 216/62/187 4 mg gelatin 10% Mg(OH)₂ Aqueous: Sonicator 20%amplitude 22 mg S/M/M (12k) 1% PVA, 5% sucrose 30 sec 20 216/62/187 2 mggelatin 10% Mg(OH)₂ Oil: Sonicator 20% amplitude No excipient (12k) 4.3mg S/M/M mineral oil + Span 80 30 sec 20 216/62/187 4 mg gelatin 10%Mg(OH)₂ Oil: Sonicator 20% amplitude No excipient (12k) mineral oil +Span 80 30 sec 20 216/62/187 4 mg gelatin 10% Mg(OH)₂ Oil: Sonicator 20%amplitude No excipient (31k) 10% Mg(OH)₂ mineral oil + Span 80 30 sec 20216/62/187 2 mg gelatin Oil: Sonicator 20% amplitude No excipient (31k)4.3 mg S/M/M mineral oil + Span 80 30 sec 20 216/62/187 4 mg gelatin 10%Mg(OH)₂, 10% Oil: Sonicator 20% amplitude No excipient (12k) myristicacid mineral oil + Span 80 30 sec 20 216/62/187 4 mg gelatin 5% Mg(OH)₂,Oil: Sonicator 20% amplitude No excipient (12k) 5% Al(OH)₃ mineral oil +Span 80 30 sec

TABLE 8 Percent D-antigen (type 1, type 3 and type 3 IPV) retained onPLA particles after air-drying for one day with the indicated excipientsand IPV at a ratio of 100:1. Polyol Other Excip:Vax Days ° C. DryingStorage Type 1 Type 2 Type 3 none Silk 100 1 25 Air-dry BSL cabinet 4 184 Maltodextrin Arginine 100 1 25 Air-dry BSL cabinet 7 84 38 MethylCellulose Ectoines 100 1 25 Air-dry BSL cabinet 62 52 57 HPMC Ubiquitin100 1 25 Air-dry BSL cabinet 59 n/a 79 HPMC MSG 100 1 25 Air-dry BSLcabinet 65 n/a 73 none Gelatin, ubiquitin 100 1 25 Air-dry BSL cabinet75 74 79 Sucrose Gelatin 100 1 25 Air-dry BSL cabinet 77 78 79 CaHeptagluconate k Phosphate 100 1 25 Air-dry BSL cabinet 78 80 86Trehalose Threonine 100 1 25 Air-dry BSL cabinet 81 83 84 CMC Gelatin100 1 25 Air-dry BSL cabinet 82 82 84 Glycerol MSG 100 1 25 Air-dry BSLcabinet 82 84 86 Ca-Heptagluconate Gelatin 100 1 25 Air-dry BSL cabinet85 n/a 83 Sorbitol Peptone 100 1 25 Air-dry BSL cabinet 84 87 85Trehalose Ubiquitin 100 1 25 Air-dry BSL cabinet 84 91 90 MannitolGlutamine 100 1 25 Air-dry BSL cabinet 91 n/a 89 Sorbitol Gylcine 100 125 Air-dry BSL cabinet 90 88 90 Maltodextrin Glutamine 100 1 25 Air-dryBSL cabinet 88 n/a 94 Maltodextrin None 100 1 25 Air-dry BSL cabinet 9395 91 Mannitol Ectoines 100 1 25 Air-dry BSL cabinet 92 94 92 TrehaloseGlycine 100 1 25 Air-dry BSL cabinet 93 90 92 Sorbitol Ectoines 100 1 25Air-dry BSL cabinet 96 n/a 92 Sucrose Threonine 100 1 25 Air-dry BSLcabinet 95 n/a 97 Sorbitol MSG, MgCl2 100 1 25 Air-dry BSL cabinet 99 9599

We claim:
 1. A polymeric device comprising a polymeric shell and atleast one discrete region comprising a therapeutic, prophylactic,nutraceutical, or diagnostic agent, optionally in combination with astabilizing excipient, wherein the shell and discrete regions are formedfrom successive layers of polymeric particles bonded together by solventand/or temperature by three dimensional printing or micromolding.
 2. Thepolymeric device of claim 1 formed by three dimensional printing ormicromolding of a biocompatible polymer, the device comprising apolymeric shell and one or more discrete regions comprising antigen,optionally in combination with a stabilizing excipient for the antigen,wherein an effective amount of the antigen is released in two or moretime periods to elicit an immune response with insufficient antigenrelease between the release periods to elicit an immune response invivo.
 3. The device of claim 2, wherein the device is made by threedimensional printing.
 4. The device of claim 2 further comprising aneffective amount of antigen to elicit an immune response in vivo whichis present in a stabilizing excipient, on the surface of the device, ormixed with the polymer of the device.
 5. The device of claim 2,comprising a stabilizing excipient selected from the group consisting ofsugars, oils, lipids, and carbohydrates.
 6. The device of claim 2,wherein the antigen elicits an immune response to an infectious agent orto a tumor.
 7. The device of claim 6, wherein the infectious agent is avirus, bacteria, fungus or protozoan.
 8. The device of claim 7, whereinthe virus is selected from the group consisting of polio, influenza,hepatitis, rotavirus, measles, mumps, rubella, and varicella.
 9. Thedevice of claim 7, wherein the bacteria is selected from the groupconsisting of Corynebacterium diphtheriae, Bordetella pertussis,Clostridium tetani, Streptococcus pneumonia (Pneumococcus), andNeisseria meningitidis (Meningococcus).
 10. The device of claim 6,wherein the antigen is a tumor antigen selectively eliciting a T cellresponse to a tumor.
 11. The device of claim 2, wherein the polymer isbiodegradable by hydrolysis.
 12. The device of claim 2 providing releaseat intervals of between ten and ninety days.
 13. The device of claim 2wherein the antigen is encapsulated in polymeric particles in the formof microparticles, microcapsules, or microspheres.
 14. The device ofclaim 2, wherein the device is injectable.
 15. The device of claim 2,wherein the device is implantable.
 16. The device of claim 2, whereinthe device can be applied to a mucosal surface selected from the groupconsisting of nasal, pulmonary, oral, vaginal and rectal.
 17. The deviceof claim 5, wherein the stabilizing excipient comprises sugar, whereinthe sugar is selected from the group consisting of sucrose, trehalose,and combinations thereof.
 18. The device of claim 2, wherein thestabilizing excipient comprises monosodium glutamate (MSG).
 19. Thedevice of claim 2, wherein the stabilizing excipient comprises magnesiumchloride (MgCl₂).
 20. The device of claim 17, wherein the sugarcomprises sucrose.
 21. The device of claim 2 providing release atintervals of between 30 and 60 days.
 22. The device of claim 2, whereinthe agent is released in at least three time periods at intervals ofbetween ten and ninety days.
 23. The device of claim 2, wherein theagent is released in at least four time periods at intervals of betweenten and ninety days.
 24. The device of claim 2, further comprising abuffering agent.
 25. The device of claim 24, wherein the buffering agentis selected from the group consisting of magnesium hydroxide, aluminumhydroxide, and myristic acid.